JP7749630B2 - Plants with altered flower color and method for producing same - Google Patents

Description

IPOD FERM BP-22483

The present invention relates to plants with altered flower color and methods for producing such plants.

The flower industry strives to develop new and diverse varieties. One effective way to develop new varieties is to change flower color, and using classical breeding methods, most commercial varieties have been developed to exhibit a variety of flower colors.

For example, Patent Document 1 has reported a method for producing plants with blue flower colors. More specifically, the method in Patent Document 1 is a method for producing plants with blue flower colors, and is characterized by causing a delphinidin-type anthocyanin, in which the 3' and 5' positions of the anthocyanin B ring are glycosylated, to coexist with a flavone glycoside or a flavonol glycoside within plant cells.

Furthermore, Patent Document 2 discloses a novel Mandevilla plant with flower colors of new tones that have not previously been produced. More specifically, the novel Mandevilla plant of Patent Document 2 is a Mandevilla plant that contains at least one type of carotenoid pigment in its petals, and is characterized in that the carotenoid pigment is neoxanthin or a derivative thereof.

International Publication No. 2017/169699 Japanese Patent Application Laid-Open No. 2021-175402

Tatsuzawa, F. etc. al. Biochem. Systemat. Ecol. 99, 2021, 104347 Macz-Pop, G. A. etc. al. Food Chem. 94, 2006, 448-456 Oyama, K. etc. al. J. Agric. Food Chem. 63, 2015, 7630-7635

There is still a need for development of plants with altered flower color.

For example, until now, only varieties of Mandevilla plants have been known with white, pink, and red flower colors, and although Patent Document 2 provides Mandevilla plants with new colors that contain carotenoid pigments, research on blue Mandevilla plants is still needed.

The present invention aims to improve the above situation, and its purpose is to provide plants with altered flower color and a method for producing such plants.

The present invention, which achieves the above objectives, is as follows:

<Aspect 1>
A method for producing a plant, comprising:
The method includes accumulating an anthocyanidin pigment in cells of petals of the plant in an amount of 30 mass% or more of the total amount of pigment present in the cells, and the flower color of the plant after accumulating the anthocyanidin pigment has changed so that the b ^* value in the CIE L ^* a ^* b ^* color system is decreased compared to the flower color of the plant before accumulating the anthocyanidin pigment.
method.
<Aspect 2>
Aspect 2. The method according to aspect 1, wherein the anthocyanidin pigment is accumulated by at least partially deleting an anthocyanidin-modifying enzyme gene.
<Aspect 3>
the method comprises deleting at least a portion of the promoter region of the anthocyanidin modifying enzyme gene, and the anthocyanidin modifying enzyme is selected from an anthocyanidin glycosidase gene and/or anthocyanidin 3-glucoside glycosidase.
The method according to aspect 2.
<Aspect 4>
the anthocyanidin glycosidase is a galactosyltransferase, and at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and sequences having at least 90% identity thereto is deleted in the promoter region of the galactosyltransferase gene;
The method according to aspect 3.
Aspect 5
the anthocyanidin 3-glucoside glycosidase is a xylosyltransferase, and at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity thereto is inserted into the promoter region of the xylosyltransferase gene.
The method according to aspect 3.
Aspect 6
Aspect 6. The method of any one of aspects 1 to 5, wherein the anthocyanidin pigment is cyanidin.
Aspect 7
Aspect 7. The method of any one of aspects 1 to 6, wherein the anthocyanidin pigment is localized in the cytoplasm of the cell.
<Aspect 8>
Aspect 8. The method according to any one of Aspects 1 to 7, wherein chlorogenic acid and aluminum ions are contained in cells of petals of the plant.
<Aspect 9>
Aspect 9. The method according to any one of Aspects 1 to 8, wherein the plant is at least one species selected from the group consisting of Apocynaceae plants, Solanaceae plants, Azollaceae plants, and Scrophulariaceae plants.
Aspect 10
Aspect 10. The method according to any one of aspects 1 to 9, wherein the plant is at least one species selected from the group consisting of plants of the genus Mandevilla, plants of the genus Petunia, and plants of the genus Torenia.
<Aspect 11>
Aspect 11. The method according to aspect 10, wherein the plant is a plant deposited at the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD) under accession number FERM ABP-22483, or a progeny thereof.
<Aspect 12>
In the cells of plant petals,
anthocyanidin pigments are accumulated in an amount of 30% by mass or more relative to the total amount of pigments present in the cells; and at least a portion of an anthocyanidin modifying enzyme gene is deleted.
plant.
<Aspect 13>
Aspect 13. The plant of aspect 12, wherein the anthocyanidin pigment is localized in the cytoplasm of the cell.
<Aspect 14>
Aspect 14. The plant according to aspect 12 or 13, comprising at least a partial deletion of a promoter region of the anthocyanidin modifying enzyme gene, and the anthocyanidin modifying enzyme is selected from anthocyanidin glycosidase and/or anthocyanidin 3-glucoside glycosidase.
Aspect 15
the anthocyanidin glycosidase is a galactosyltransferase, and at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and sequences having at least 90% identity thereto is deleted in the promoter region of the galactosyltransferase gene;
A plant according to aspect 14.
<Aspect 16>
the anthocyanidin 3-glucoside glycosidase is a xylosyltransferase, and at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity thereto is inserted in the promoter region of the xylosyltransferase gene;
A plant according to aspect 14.
Aspect 17
Aspect 17. The plant according to any one of aspects 12 to 16, wherein the anthocyanidin pigment is cyanidin.
<Aspect 18>
Aspect 18. The plant according to any one of aspects 12 to 17, wherein chlorogenic acid and aluminum ions are contained in cells of petals of the plant.
<Aspect 19>
Aspect 19. The plant according to any one of aspects 12 to 18, wherein the plant is at least one species selected from the group consisting of Apocynaceae plants, Solanaceae plants, Azollaceae plants, and Scrophulariaceae plants.
<Aspect 20>
Aspect 20. The plant according to any one of aspects 12 to 19, wherein the plant is at least one species selected from the group consisting of plants of the genus Mandevilla, plants of the genus Petunia, and plants of the genus Torenia.
<Aspect 21>
Aspect 21. The plant according to aspect 20, wherein the Mandevilla plant is a plant deposited at the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD) under accession number FERM ABP-22483, or a progeny thereof.
<Aspect 22>
A progeny of the plant according to any one of aspects 12 to 21.
<Aspect 23>
22. Propagation material of a plant according to any one of aspects 12 to 21.
<Aspect 24>
A part, tissue or cell of the plant according to any one of aspects 12 to 21.

The present invention makes it possible to provide plants with altered flower color.

FIG. 1 shows the results of pigment analysis in Example 1. FIG. 2 shows the results of HPLC analysis in Example 1. FIG. 3 is a diagram showing the results of the structural analysis in Example 2. FIG. 4 shows the expression analysis of the galactosyltransferase gene and the xylosyltransferase gene in Example 3. FIG. 5 shows the results of LC-MS analysis in Example 4. FIG. 6 shows the results of extraction using various solvents in Example 5. FIG. 7 shows the experiment of Example 6. FIG. 8 shows the results of analysis of the nucleotide sequence of the galactosyltransferase gene in Example 7 and the alignment. FIG. 9 shows the analysis results and alignment of the base sequences of xylosyltransferase genes in Example 7. FIG. 10 shows a design for creating a genetic marker in Example 7. FIG. 11 shows the results of PCR amplification in Example 7. FIG. 12 is a diagram showing the intracellular localization of the dye in Example 8.

The following describes in detail the embodiments of the present invention. Note that the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the invention.

<<Plant Creation Method>>
The method for producing a plant of the present invention (hereinafter also referred to simply as the "method of the present invention") comprises:
The method includes accumulating an anthocyanidin pigment in the cells of the petals of the plant in an amount of 30 mass% or more of the total amount of pigment present in the cells, and the flower color of the plant after accumulating the anthocyanidin pigment changes so that the b ^* value in the CIE L ^* a ^* b ^* color system decreases compared to the flower color of the plant before accumulating the anthocyanidin pigment.
It is a method.

As described in Patent Document 1 above, there has been a technical idea of accumulating glycoside-form anthocyanin pigments and the like within petal cells. Furthermore, it is known that aglycone-form pigments are generally unstable and become stabilized within cells through modifications such as glycosylation and acylation. Therefore, there have been no reports of the technical idea of accumulating anthocyanidin pigments, i.e., aglycone-form pigments, within petal cells, nor have there been any reports of anthocyanidin pigments accumulating within the petal cells of plants.

For this reason, the technical concept of the present invention, of accumulating anthocyanidin pigments in an amount of 30% by mass or more of the total amount of pigments present in the petal cells of a plant, is novel, and the results of Examples 1 to 8 described below suggest that the method of the present invention makes it possible to accumulate anthocyanidin pigments in the petal cells (particularly in the cytoplasm) of a plant.

According to the method of the present invention, anthocyanidin pigments can be accumulated in plant petal cells at a concentration of 30% by mass or more relative to the total amount of pigment present in the cells. More specifically, this accumulated amount of anthocyanidin pigments can be 30% by mass or more, 35% by mass or more, 40% by mass or more, 45% by mass or more, 50% by mass or more, 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 95% by mass or more, 99% by mass or more, or approximately 100% by mass relative to the total amount of pigment present in the cells. The accumulated amount of anthocyanidin pigments and the total amount of pigment present in the cells can be determined, for example, by extracting the pigments from freeze-dried petals with a mixture of trifluoroacetic acid and acetonitrile and performing HPLC analysis. For more specific details, see Examples 1 and 2 described below.

In addition, in the present invention, the amount of anthocyanidin pigments in a plant after accumulation of the anthocyanidin pigments is not particularly limited, and may be, for example, 0.05 mg or more, 0.1 mg or more, 0.2 mg or more, 0.3 mg or more, 0.4 mg or more, 0.5 mg or more, 0.6 mg or more, 0.7 mg or more, 0.8 mg or more, 0.9 mg or more, 1.0 mg or more, 1.0 mg or more, 1.2 mg or more, 1.3 mg or more, 1.4 mg or more, 1.5 mg or more, or 2.0 mg or more per 1 g of petals, or 2.5 mg or less, 2.0 mg or less, 1.8 mg or less, 1.5 mg or less, or 1.1 mg or less.

In the present invention, the index of change in flower color of a plant before and after accumulation of an anthocyanidin pigment can be determined by the b ^* value in the CIE L ^* a ^* b ^* color system. More specifically, it is sufficient that the flower color of a plant after accumulation of anthocyanidin pigments has changed so that the b ^* value in the CIE L ^* a ^* b ^* color system is reduced compared to the flower color of the plant before accumulation of anthocyanidin pigments.

Here, the CIE L ^* a ^* b ^* color system is a color system standardized by the International Commission on Illumination (CIE) and is widely known as a notation method for expressing color tones. It is also called the CIE 1976 (L ^* a ^* b ^* ) color system. In the L ^* a ^* b ^* color system, lightness is represented by L ^* , and chromaticity, which indicates hue and saturation, is represented by a ^* and b ^* . a ^* and b ^* indicate the color direction, with a ^* representing the red direction, -a ^* representing the green direction, b ^* representing the yellow direction, and -b ^* representing the blue direction. The ^L* , a ^* , and b ^* values can be obtained from the tristimulus values X, Y, and Z using the following equations:
L ^* = 116 (Y/Y ₀ ) ^1/3 - 16;
a ^* = 500 [(X/X ₀ ) ^1/3 - (Y/Y ₀ ) ^1/3 ]; and b ^* = 200 [(Y/Y ₀ ) ^1/3 - (Z/Z ₀ ) ^1/3 ]
However, X/X _{0 ,} Y/Y ₀ and Z/Z ₀ are >0.008856, where X ₀ , Y ₀ and Z ₀ represent the tristimulus values of the standard illuminant.

Furthermore, color analysis according to the CIE L ^* a ^* b ^* color system can be easily performed using an integrating sphere type spectrocolorimeter, for example, a commercially available spectrophotometer (CM-2022, Konica Minolta).

According to the method of the present invention, a decrease in the b ^* value means that the flower color of the plant after accumulation of anthocyanidin pigments shifts toward a bluer shade. The degree of decrease in the b ^* value is not particularly limited and may be, for example, 5.0 or more, 10.0 or more, 15.0 or more, 20.0 or more, 25.0 or more, 30.0 or more, 35.0 or more, 40.0 or more, 45.0 or more, or 50.0 or more.

Furthermore, in the present invention, the b ^* value of the flower color of the plant after accumulation of anthocyanidin pigments is not particularly limited, and may be, for example, 20.0 or less, 15.0 or less, 10.0 or less, 5.0 or less, 1.0 or less, or 0 or less. The b ^* value may preferably be less than 0. Furthermore, when the b ^* value is less than 0, more specifically, its absolute value may be, for example, 1.0 or more, 5.0 or more, 10.0 or more, 15.0 or more, 20.0 or more, 25.0 or more, or 30.0 or more.

In one embodiment, according to the method of the present invention, when a Mandevilla plant with red flower color is used, the flower color of the Mandevilla plant after accumulation of anthocyanidin pigments changes as the b* value in the CIE L ^* a ^* b ^* color system decreases from "20.9" to "-30.9", compared to the flower color ( ^red ) of the Mandevilla plant before accumulation of anthocyanidin pigments, i.e., the flower color can be shifted toward the blue side.

Plants that can be used in the method of the present invention are not particularly limited and may be, for example, at least one species selected from the group consisting of plants of the Apocynaceae family, the Solanaceae family, the Linderniaceae family, and the Scrophulariaceae family.

Furthermore, plants that can be used in the method of the present invention may be at least one species selected from the group consisting of plants of the genus Mandevilla, the genus Petunia, and the genus Torenia, but are not limited to these.

It is generally known that the red, blue, and purple flower colors found in plants are derived from anthocyanin pigments. Anthocyanin pigments are formed when sugars are added to anthocyanidin (aglycone) pigments using glycosyltransferases, etc.

In the present invention, an anthocyanidin pigment is an aglycone of an anthocyanin pigment, and has the structure of a compound represented by the following general formula (I):
(In formula (I), R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ may each independently represent a hydrogen atom, an —OH group, or an —OCH ₃ group.)

In general formula (I), anthocyanin pigments are classified into three types: pelargodinin, cyanidin, and delphinidin, depending on the number of hydroxy groups on the B ring of the anthocyanidin moiety. Furthermore, various anthocyanin pigments can exist depending on the type and number of sugars added to the A ring and C ring of the anthocyanidin moiety.

In the method of the present invention, the anthocyanidin pigment may be cyanidin, which has the structure shown in formula (II):

When using the method of the present invention, for example, with a Mandevilla plant whose flowers are red, it is preferable to accumulate cyanidin in the petal cells of the plant. This allows the flower color of the Mandevilla plant after cyanidin accumulation to change to blue, compared to the flower color (red) of the Mandevilla plant before cyanidin accumulation.

In the present invention, anthocyanidin pigments may be localized in the cytoplasm of plant petal cells, and in particular, may be localized in the cytoplasm of plant petal epidermal cells. In contrast, anthocyanin pigments are generally localized in vacuoles in petal cells. The intracellular location of the pigment (i.e., the location where it is present) can be confirmed, for example, by preparing protoplasts from petals and observing them under a phase-contrast microscope using a hemocytometer slide.

In the method of the present invention, anthocyanidin pigments can be accumulated in plant petal cells by at least partially deleting an anthocyanidin-modifying enzyme gene. Here, the anthocyanidin-modifying enzyme may be an enzyme that can modify anthocyanidins in the aglycone state, such as an enzyme that glycosylates anthocyanidins or an enzyme that acylates anthocyanidins.

In one aspect of the present invention, the anthocyanidin-modifying enzyme may be selected from anthocyanidin glycosidase and/or anthocyanidin 3-glucoside glycosidase. In this case, in the method of the present invention, anthocyanidin pigments can be accumulated by at least partially deleting the anthocyanidin glycosidase gene and/or the anthocyanidin 3-glucoside glycosidase gene. Preferably, anthocyanidin pigments can be accumulated by at least partially deleting both the anthocyanidin glycosidase gene and the anthocyanidin 3-glucoside glycosidase gene.

Without being limited by theory, for example, by at least partially deleting the anthocyanidin glycosidase gene and the anthocyanidin 3-glucoside glycosidase gene in the cells of plant petals, anthocyanidin pigments cannot be converted to their glycosides, anthocyanins, and as a result, anthocyanidin pigments may accumulate. The sites of deletion in the anthocyanidin glycosidase gene and the anthocyanidin 3-glucoside glycosidase gene are not particularly limited and may be, for example, the respective promoter regions.

Furthermore, deletion of the anthocyanidin modifying enzyme gene may be achieved using conventional techniques in the art, such as mutagenesis using irradiation with gamma rays or heavy ion beams, RNAi, genome editing, etc. More specifically, deletion may be achieved by deleting, inserting, substituting, and/or adding at least a portion of the DNA sequence in the anthocyanidin modifying enzyme gene.

In the method of the present invention, the anthocyanidin glycosidase may specifically be, for example, a galactosyltransferase. The galactosyltransferase gene may have the DNA sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 5. Furthermore, the anthocyanidin 3-glucoside glycosidase may specifically be, for example, a xylosyltransferase. The xylosyltransferase gene may have the DNA sequence shown in SEQ ID NO: 6 and the amino acid sequence shown in SEQ ID NO: 7.

For example, when cyanidin is treated with galactosyltransferase and xylosyltransferase, the cyanidin can undergo a two-step saccharification reaction to become the cyanidin glycoside shown in formula (III) below. In the first step, galactose is added to the 3-position of cyanidin by the galactosyltransferase, forming cyanidin 3-galactoside. Then, in the second step, xylose can be further added to cyanidin 3-galactoside by the xylosyltransferase.

(In formula (III), Gal represents galactose, and Xyl represents xylose.)

Furthermore, in the method of the present invention, when the anthocyanidin glycosidase is a galactosyltransferase, at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and sequences having at least 90% identity thereto may be deleted in the promoter region of the galactosyltransferase gene.

In addition, to identify whether a galactosyltransferase gene has a deleted sequence, PCR primers may be designed to span the deleted portion, compared to a gene without a deleted sequence. Then, PCR is performed using the designed primers, and if an amplified fragment of the desired length is obtained, the gene can be identified as having a deleted sequence.

In the method of the present invention, when the anthocyanidin 3-glucoside glycosidase is a xylosyltransferase, at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 3 and sequences having at least 90% identity thereto may be inserted into the promoter region of the xylosyltransferase gene.

In addition, to identify whether a sequence has been inserted into a xylosyltransferase gene, PCR primers may be designed from the inserted portion, for example, compared to a gene without an inserted sequence. Then, PCR is performed using the designed primers, and if an amplified fragment of the desired length is obtained, the gene can be identified as having an inserted sequence.

In the present invention, the term "identity" refers to the amount (number) of amino acid residues or bases that can be determined to be identical between two chains in a polypeptide sequence (or amino acid sequence) or polynucleotide sequence (or base sequence) in terms of their matching relationship with each other, and refers to the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences, and "identity" can be easily calculated. Numerous methods for determining identity between two polynucleotide or polypeptide sequences are known, and the term "identity" is well known to those skilled in the art (see, e.g., Lesk, A. M. (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, D. W. (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, A. M. & Grifin, H. G. (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G. , Sequence Analysis in Molecular Biology, Academic Press, New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.).

Unless otherwise specified, the "identity" values described herein may be values calculated using identity search programs known to those skilled in the art, but are preferably values calculated using the ClustalW program in the MacVector application (version 9.5, Oxford Molecular Ltd., Oxford, England). In the present invention, the degree of "identity" between each sequence may be, for example, about 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, particularly preferably 98% or more, and most preferably 99% or more.

In the present invention, chlorogenic acid and aluminum ions may be contained in the petal cells of the plant. The presence of chlorogenic acid and aluminum ions can result in a more pronounced change in flower color due to the co-pigment effect. For example, when a Mandevilla plant with red flowers is used, the presence of chlorogenic acid and aluminum ions in the petal cells of the plant can result in a more pronounced change in flower color to blue after cyanidin accumulation compared to the flower color (red) of the Mandevilla plant before cyanidin accumulation.

"plant"
The present invention also provides a plant. The plant of the present invention comprises:
In the cells of plant petals,
anthocyanidin pigments are accumulated in an amount of 30% by mass or more relative to the total amount of pigments present in the cells, and at least a portion of the anthocyanidin modifying enzyme gene is deleted.
It is a plant.

The plant of the present invention may be produced by the method of the present invention described above. Therefore, for an explanation of the anthocyanidin-modifying enzyme, the description of the method of the present invention described above may be referred to as appropriate.

In addition, in the plant of the present invention, at least a portion of the promoter region of the anthocyanidin modifying enzyme gene may be deleted. The anthocyanidin modifying enzyme may be selected from anthocyanidin glycosidase and/or anthocyanidin 3-glucoside glycosidase. Furthermore, when the anthocyanidin glycosidase is a galactosyltransferase, at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and sequences having at least 90% identity thereto may be deleted in the promoter region of the galactosyltransferase gene. Furthermore, when the anthocyanidin 3-glucoside glycosidase is a xylosyltransferase, at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 3 and sequences having at least 90% identity thereto may be inserted in the promoter region of the xylosyltransferase gene.

The anthocyanidin pigment of the plant of the present invention can be localized in the cytoplasm of the cells of the plant's petals. Furthermore, in the plant of the present invention, the anthocyanidin pigment may be cyanidin.

The plant of the present invention may be at least one species selected from the group consisting of plants of the Apocynaceae family, Solanaceae family, Azollaceae family, and Scrophulariaceae family, and more specifically, may be at least one species selected from the group consisting of plants of the Mandevilla genus, Petunia genus, and Torenia genus.

The Mandevilla plant of the present invention can be produced from a Mandevilla plant deposited with the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD) under accession number FERM ABP-22483, or its progeny, using conventional plant breeding techniques. Such conventional techniques may include conventional propagation techniques such as vegetative propagation and seed propagation, as well as tissue culture techniques for culturing plant cells, tissues, or organs, and genetic recombination techniques that can directly introduce useful traits. The Mandevilla plant of the present invention can typically be produced by vegetative propagation of a Mandevilla plant or its progeny deposited with the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD) under accession number FERM ABP-22483 on September 22, 2023. Vegetative propagation can be achieved by methods such as cuttings (including leaf cuttings), layering, stem laying (pressing method), division, grafting, and even cloned seedlings created by tissue culture, but cuttings are particularly preferred.

"Cutting" is an asexual propagation method in which a part of a plant that does not have a stem or roots is cut off and trained to become an independent plant with a stem and roots. Depending on the part used, cuttings can be branch cuttings, trunk cuttings (stem cuttings), root cuttings, leaf cuttings, etc. Cuttings are typically performed by inserting a part of the branch or trunk (stem) of the parent plant into soil and leaving it partly above ground. In the present invention, there are no particular limitations on the part used for "cutting."

Additionally, cuttings may be taken from a Mandevilla plant or its progeny, which has been deposited with the National Institute of Technology and Evaluation's International Patent Organism Depositary (NITE-IPOD) under accession number FERM ABP-22483. "Mutation" refers to a phenomenon in which flowers, buds, leaves, etc., on only a certain branch of a plant, develop traits that differ from the genetic traits of the individual due to a mutation in the meristem or other factors, or to an individual that exhibits such a phenomenon. As the plant develops from the meristem forward, this trait may be differentiated from the unmutated portion and become fixed. Therefore, by taking cuttings from a branch of a mutant meristem bearing at least one altered flower, it is possible to create a new Mandevilla plant with other traits while maintaining a desired trait (e.g., blue flower color).

In another aspect, the present invention provides propagation materials (vegetative propagation materials such as cuttings, seeds, etc.), plant parts (flowers, stems, branches, leaves, roots, etc.), tissues, or cells of the above-mentioned Mandevilla plant or its progeny.

In a further aspect, the present invention provides a product derived from the Mandevilla plant or its progeny, typically a potted plant, a cut flower, or a processed product made from the cut flower.

As used herein, "progeny" includes self- or cross-pollinated progeny of Mandevilla plants deposited with the National Institute of Technology and Evaluation (NITE-IPOD) under accession number FERM ABP-22483, and includes not only the first generation but all generations of progeny of Mandevilla plants deposited with the National Institute of Technology and Evaluation (NITE-IPOD) under accession number FERM ABP-22483. Furthermore, as used herein, the term "progeny" includes those derived from a parent Mandevilla plant based on conventional techniques in plant breeding. For example, progeny may include those produced by conventional breeding techniques, such as vegetative propagation and seed propagation of a parent plant, as well as tissue culture techniques for cultivating plant cells, tissues, or organs, and recombinant DNA techniques that can directly introduce useful traits, as well as mutants such as branch mutations of a parent Mandevilla plant. Such offspring preferably have the desired characteristics derived from the parent Mandevilla plant.

The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

Example 1
In Example 1, the pigment compositions contained in the petal cells of a blue mandevilla cultivar (MW65) with blue flower color, a red mandevilla cultivar (173) with red flower color, and a hybrid (13918) of these mandevillas, all of which were owned by Suntory Flowers Ltd., were analyzed by HPLC. Here, the hybrid (13918) was a cross between the blue cultivar (MW65) as the mother and a cultivar from a different lineage than the red cultivar (173) as the father.

The color analysis of each of the above varieties (blue variety, red variety, and their hybrids) according to the CIE L ^* a ^* b ^* color system was carried out using a commercially available spectrophotometer (CM-2022, Konica Minolta). The results are shown in Table 1 below.

Non-Patent Document 1 reports that cyanidin is the predominant anthocyanidin in the petal cells of plants of the genus Mandevilla. Therefore, the anthocyanin pigments in the petals of each of the above varieties (blue variety, red variety, and their hybrids) were analyzed as follows.

(Test solution preparation)
4 mL of 0.1% TFA/50% acetonitrile was added to 0.5 mg of each freeze-dried petal, and anthocyanin pigments were extracted under ultrasound for 20 minutes.
The glycosides were analyzed by HPLC as they were in the extract.
In the case of analyzing the aglycones from which sugars and other components had been removed, the extract was dried overnight in a desiccator, dissolved in 6N HCl, saponified in a boiling water bath for 20 minutes, extracted with amyl alcohol, and then analyzed by HPLC.

(HPLC analysis)
The analytical equipment used was a Shimadzu Prominence HPLC system (LC-20AD, SIL-20AC, CBM-20A, CTO-20AC, SPD-M20A, SPD-20A).
For glycoside analysis, an RSpak DE-413L (250 mm x 4.6 mm ID, Shodex) column was used, and the LC mobile phases used were 0.5% TFA/water for mobile phase A and 0.5% TFA-containing 50% CH3CN/water for mobile phase B. The binary gradient gradient was changed from 20% to 100% over 15 minutes, and then the mobile phase B concentration was changed to 100% for 10 minutes at a flow rate of 0.4 mL/min.
For the analysis of aglycones, a YMC-Pack ODS-A column (150 mm x 6 mm ID, YMC) was used, and the LC mobile phase was acetic acid/methanol/water (15/17.5/67.5, vol/vol), at a flow rate of 1.0 mL/min.
The peaks were identified and quantified using cyanidin, delphinidin, pelargonidin, malvidin, peonidin, and petunidin as standard substances.

As a result, as shown in Figure 1, it was found that cyanidin was the major anthocyanidin in both the blue variety (MW 65) and the red variety (173) in samples from which sugars had been removed by acid hydrolysis. This result is also consistent with the report in Non-Patent Document 1.

Furthermore, as shown in Figure 2, analysis of the sugared varieties detected specific high peaks (Peak A and Peak B) in the red variety (173) and the blue variety (MW 65). Both peaks (Peak A and Peak B) were detected in the hybrid variety (13918). In Example 2 below, structural analysis was performed on these peaks.

Example 2
The structures of peaks A and B in the red variety (173) and blue variety (MW 65) in Example 1 above were estimated by LC-MS.

More specifically, petal extracts were prepared using 5% AcOH, 50% MeCN-5% AcOH, and MeCN-5% AcOH. LC-MS experiments were performed using an LCMS-IT-TOF (Shimadzu) column and a YMC-Triart C18 (3 μm, 2.1 x 100 mm, YMC) column. The LC mobile phases used were 0.1% formic acid/water (mobile phase A) and 0.1% formic acid/acetonitrile (mobile phase B). A binary gradient gradient was used, varying the mobile phase B concentration from 20% to 100% over 15 minutes, followed by 10 minutes at 100% mobile phase B, at a flow rate of 0.4 mL/min. Anthocyanin peaks were extracted from the chromatogram at 530 nm, and structural analysis was performed based on MS and MS/MS spectra.

As a result, as shown in Figure 3, peak A of the red variety (173) was found to be cyanidin 3-O-[2-O-(xylosyl)-galactoside], as described in Non-Patent Document 1.

On the other hand, surprisingly, peak B from the blue variety (MW 65) was found to be completely unmodified cyanidin (aglycone) = anthocyanidin (Figure 3). There were almost no known examples of aglycones accumulating in large amounts within plants, so this analytical result overturned conventional wisdom.

Furthermore, Non-Patent Document 2 reports that in some kidney bean varieties, anthocyanidins accumulate in the pericarp, but the percentage is only 22%. In contrast, it was found that cyanidin, shown by Peak B, accumulates at 70% by mass or more of the total amount of pigment in the cytoplasm and vacuoles of petal cells of the blue variety (MW65).

Example 3
In the blue cultivar (MW65), we believe that high accumulation of anthocyanidins is due to reduced expression or enzymatic activity in the petals of the galactosyltransferase gene and xylosyltransferase gene, which are thought to be necessary for synthesizing cyanidin 3-O-[2-O-(xylosyl)-galactoside] from cyanidin aglycone.

First, we identified the galactosyltransferase and xylosyltransferase genes in Mandevilla as follows. Glycosyltransferase genes were compiled from the gene cluster deduced from the Mandevilla genome sequence, and the amino acid sequences of the proteins they encoded were aligned with previously reported anthocyanidin glycosyltransferase sequences using ClustalW, followed by a phylogenetic tree created using ETE3. From the resulting phylogenetic tree, we inferred genes that are likely orthologs of previously reported galactosyltransferase and xylosyltransferase genes in other plants. Expression analysis of these gene clusters was performed using RNA-seq and RT-PCR.

(RNA-seq method)
After pretreatment with Fruit-mate for RNA Purification (Takara Bio), total RNA was extracted from mandevilla petals using RNeasy Plant mini (Qiagen). An RNA-Seq library was prepared using the MGI Easy RNA Directional Library Prep Set (MGI). Paired-end sequence data of 150 bases was obtained using the MGI DNBSeq-G400RS. After removing low-quality sequences and adapter sequences, the sequence was mapped to a reference sequence to calculate expression levels.

(Quantitative RT-PCR method)
The total RNA was subjected to reverse transcription using ReverTra Ace (Toyobo) to prepare cDNA. Quantitative PCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) and the StepOnePlus real-time PCR system (Applied Biosystems). Expression levels were calculated using the actin gene as an endogenous control.

As a result, as shown in Figure 4, both genes (galactosyltransferase gene and xylosyltransferase gene) were highly expressed in the red cultivar (173), but their expression was barely observed in the blue cultivar (MW65). Furthermore, these results are consistent with the idea that in blue mandevilla, the majority of cyanidin exists in the aglycone form without being modified with sugars.

Example 4
Aglycones are generally unstable and are known to be stabilized intracellularly by modifications such as glycosylation and acylation. One possibility for the stabilization of highly accumulated cyanidin aglycones is the copigment effect (see, for example, Non-Patent Document 3). The copigment effect is well known in hydrangeas, where anthocyanins combine with substances such as flavones (which are mostly colorless or pale yellow) and metal ions in vivo to enhance the blue color. A blue cultivar (MW 65) was searched for substances that may act as one of the causes of blue flower development using LC-MS.

(LC-MS)
Petal extracts were prepared using 5% AcOH, 50% MeCN-5% AcOH, and MeCN-5% AcOH. LC-MS experiments were performed using an LCMS-IT-TOF (Shimadzu) column on a YMC-Triart C18 (3 μm, 2.1 x 100 mm, YMC) column. The LC mobile phases were 0.1% formic acid/water (mobile phase A) and 0.1% formic acid/acetonitrile (mobile phase B). A binary gradient gradient was run over 15 minutes, varying the mobile phase B concentration from 20% to 100%, followed by 10 minutes at 100% mobile phase B, at a flow rate of 0.4 mL/min. A large peak was observed in the 325 nm chromatogram at approximately 6.3 minutes for blue-hued Mandevilla species. Structural analysis of this peak was performed based on MS and MS/MS spectra.

As a result, as shown in Figure 5, it was found that chlorogenic acid (3-CQA) accumulated at high concentrations in the blue variety (MW65). This suggests that chlorogenic acid (3-CQA) may contribute to the blue color of the blue variety (MW65).

Example 5
When 2 g of blue mandevilla flower petals were squeezed using a garlic press, a red juice was obtained at pH 3.7 (see Figure 6(a)). This result suggests that cyanidin aglycone exhibits red color at low pH and that cyanidin aglycone must be localized in the cytoplasm to produce the blue color.

Then, when the petals of the blue variety of mandevilla were treated with various solvents, all of the pigment was extracted into the methanol when extracted with amphiphilic methanol, turning the petals white and revealing that all of the pigment had been extracted (see Figure 6 (b)). This is thought to be because both the cyanidin aglycone and glycoside were extracted, and the methanol containing the pigment turned blue.

On the other hand, when extraction was performed using the organic solvent hexane (hydrophobic), the hexane remained colorless and transparent, but the petals turned red (see Figure 6 (c)). This is thought to be because the fat-soluble aglycone was extracted, but the glycoside remained, causing the petals to turn red.

Example 6
In vitro pigment reconstitution experiments were conducted to observe color development under neutral conditions simulating the cytoplasmic environment. Cyanidin chloride (ChromaDex) and 3-O-caffeoylquinic acid (Nagara Science) were dissolved in DMSO to a concentration of 10 mM. Reconstitution experiments using cyanidin alone were performed by adding the above cyanidin chloride solution to 50 mM phosphate buffer at pH 4.4 to 9.3 to a concentration of 0.5 mM, mixing, and allowing the reaction to proceed at room temperature. Reconstitution experiments using copigment and metal ions were performed by adding the above cyanidin chloride solution and 3-O-caffeoylquinic acid solution to 50 mM phosphate buffer at pH 4.4 to 9.3 to a concentration of 0.5 mM, adding aluminum ion solution (1 mM ammonium aluminum sulfate solution) to an aluminum ion concentration of 0.1 mM, and allowing the reaction to proceed at room temperature (see the table in Figure 7).

As a result, it was observed that cyanidin aglycone alone produced a blue color at neutral pH (see Figure 7). This strongly suggests that when cyanidin aglycone is present in the cytoplasm, it can produce a blue color on its own without the action of copigments or metal ions.

Example 7
To create a marker specific to the blue variety (MW65), the nucleotide sequences of the galactosyltransferase gene and xylosyltransferase gene and their neighboring regions were analyzed. The results revealed that two deletions of 16 bp and 30 bp were present in the promoter region of the galactosyltransferase gene, and a 173 bp insertion in the promoter region of the xylosyltransferase gene were specifically present in the blue variety (see Figures 8 and 9). Two sequences of paternal and maternal origin were obtained from the red variety (173), and these were named 173-h1 and 173-h2, respectively. Therefore, the galactosyltransferase gene of the red variety 173-h1 has the sequence shown in SEQ ID NO: 8, and the galactosyltransferase gene of the red variety 173-h2 has the sequence shown in SEQ ID NO: 9. Furthermore, the xylosyltransferase gene of the red variety 173-h1 has the sequence shown in SEQ ID NO: 10, and the xylosyltransferase gene of the red variety 173-h2 has the sequence shown in SEQ ID NO: 11.

For the galactosyltransferase gene, PCR primers were designed to span this deletion region (g21571-F7 (SEQ ID NO: 12): ACTGGCCTCGCCATTAACG, g21571-R5 (SEQ ID NO: 13): GTAATTTAATTAAGATGCGTAATTCTCTG) (see Figure 10).

For the xylosyltransferase gene, PCR primers were designed within this insert (g19028-F7 (SEQ ID NO: 14): AGTGTCTCTTCCTTAGTTCCTG, g19028-R5 (SEQ ID NO: 15): CTGAATTTTAAGAGATCGTTCATAATACG) (see Figure 10).

DNA extracted from leaves of the blue variety (MW65), red variety (173), and hybrid variety (13918) using NucleoSpin Plant II (MACHEREY-NAGEL) was used as a template for PCR using the primers described above. PCR was performed using Tks gflex DNA polymerase (Takara Bio) under the following conditions: 98°C for 15 seconds, 60°C for 20 seconds, and 68°C for 30 seconds, for a total of 30 cycles.

As a result, a 122 bp amplified fragment was obtained for the galactosyltransferase gene in the blue cultivar (MW65) and hybrid (13918), whereas no amplification was observed in the red cultivar (173) (see Figure 11(a)). A 121 bp amplified fragment was obtained for the xylosyltransferase gene in the blue cultivar (MW65) and hybrid (13918), whereas no amplification was observed in the red cultivar (173) (see Figure 11(b)).

In this way, by performing PCR using these primers and examining whether an amplified fragment of the desired length is obtained, it was found that it is possible to easily distinguish between the blue variety (MW65) and its progeny.

Example 8
To confirm the intracellular localization of the pigment, protoplasts were prepared from petals of the blue cultivar (MW65), red cultivar (173), and hybrid cultivar (13918), and the location of the pigment was confirmed.

Protoplasts were prepared as follows: 0.1 g of petal fragments and 10 mL of enzyme solution (1% (w/v) Cellulase "ONOZUKA" RS (Yakult Pharmaceutical Industry), 1% (w/v) Mecerozyme (Yakult Pharmaceutical Industry), 180 mM KCl, 20 mM CaCl2, 20 mM MgCl2, pH 5.5) were added to a 100 mL Erlenmeyer flask and incubated for 90 minutes in an incubator at 30°C with reciprocal shaking at 40 mm amplitude and 50 strokes per minute. The protoplasts were then transferred to a 50 mL round-bottom glass centrifuge tube and centrifuged at 100 × g (730 rpm) for 2 minutes to precipitate the protoplasts. The precipitate was then gently suspended in the remaining liquid using a pipette. The suspension was added to 10 mL of 0.5 M mannitol and used as a sample for observation. The sample was placed on a hemocytometer slide and observed under a phase-contrast microscope BX40 (Olympus).

In the red cultivar (173), the red pigment was uniformly distributed throughout the vacuoles (see Figure 12 (a) and (b)), whereas in the blue cultivar (MW65), the cytoplasm, rather than the vacuoles, was observed to be stained blue, and blue granular structures were also occasionally observed (see Figure 12 (e) and (f)).

Both states were observed in the hybrid (13918), with vacuoles stained uniformly red, while blue granular structures were observed localized in the cytoplasm (see Figure 12 (c) and (d)). This revealed that cyanidin aglycone is localized in the cytoplasm, rather than in the vacuoles where glycosides are localized, and that it forms granular structures in some areas. This localization of the pigment in the cytoplasm is a previously unknown phenomenon, and is thought to be a trait that characterizes the blue variety (MW65) and its progeny.

Claims (13)

A method for producing a plant, comprising:
the method comprises accumulating an anthocyanidin pigment in cells of petals of the plant in an amount of 30% by mass or more relative to the total amount of pigment present in the cells, and the flower color of the plant after accumulating the anthocyanidin pigment has changed so that the b ^* value in the CIE L ^* a ^* b ^* color system is decreased compared to the flower color of the plant before accumulating the anthocyanidin pigment,
The method includes at least partially deleting an anthocyanidin modifying enzyme gene to accumulate the anthocyanidin pigment,
The method comprises:
The method comprises deleting at least a portion of the promoter region of the anthocyanidin modifying enzyme gene; and
the anthocyanidin-modifying enzyme is selected from anthocyanidin glycosidase and/or anthocyanidin 3-glucoside glycosidase;
the anthocyanidin glycosidase is a galactosyltransferase; and
In the promoter region of the galactosyltransferase gene, at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and sequences having at least 90% identity thereto is deleted, the anthocyanidin 3-glucoside glycosidase is a xylosyltransferase, and
Inserting at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity thereto in the promoter region of the xylosyltransferase gene;
The plant is a Mandevilla plant.
method. 2. The method of claim 1 , wherein the anthocyanidin pigment is cyanidin. The method of claim 1 or 2, wherein the anthocyanidin pigment is localized in the cytoplasm of the cell. The method of claim 1 or 2, wherein chlorogenic acid and aluminum ions are contained in the petal cells of the plant. The method according to claim 1, wherein the Mandevilla plant is a plant having accession number FERM BP-22483 deposited at the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD ) under accession number FERM ABP-22483, or a progeny thereof. In the cells of plant petals,
anthocyanidin pigments are accumulated in an amount of 30% by mass or more of the total amount of pigments present in the cells; and at least a portion of an anthocyanidin modifying enzyme gene is deleted .
The method comprises deleting at least a portion of the promoter region of the anthocyanidin modifying enzyme gene; and
the anthocyanidin-modifying enzyme is selected from anthocyanidin glycosidase and/or anthocyanidin 3-glucoside glycosidase;
the anthocyanidin glycosidase is a galactosyltransferase; and
In the promoter region of the galactosyltransferase gene, at least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2, and sequences having at least 90% identity thereto is deleted;
the anthocyanidin 3-glucoside glycosidase is a xylosyltransferase; and
At least one sequence selected from the group consisting of the sequence shown in SEQ ID NO: 3 and a sequence having at least 90% identity thereto is inserted in the promoter region of the xylosyltransferase gene,
The plant is a Mandevilla plant .
plant. The plant of claim 6 , wherein the anthocyanidin pigment is localized in the cytoplasm of the cell. The plant of claim 6 , wherein the anthocyanidin pigment is cyanidin. The plant according to claim 6 , wherein chlorogenic acid and aluminum ions are contained in the cells of the petals of the plant. the Mandevilla plant is a plant having accession number FERM BP-22483, which has been deposited at the National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD) under accession number FERM ABP-22483, or a progeny thereof;
In the progeny, anthocyanidin pigments are accumulated in the cells of the petals of the plant in an amount of 30% by mass or more of the total amount of pigments present in the cells, and at least a portion of the anthocyanidin modifying enzyme gene is deleted.
The plant according to claim 6 . A progeny of the plant according to any one of claims 6 to 10 ,
In the progeny, anthocyanidin pigments are accumulated in the cells of the petals of the plant in an amount of 30% by mass or more of the total amount of pigments present in the cells, and at least a portion of the anthocyanidin modifying enzyme gene is deleted.
descendants. A propagation material of the plant according to any one of claims 6 to 10 ,
In the plant, anthocyanidin pigments are accumulated in the cells of the petals of the plant in an amount of 30% by mass or more of the total amount of pigments present in the cells, and at least a portion of anthocyanidin modifying enzyme genes is deleted.
Breeding material. A part, tissue or cell of the plant body according to any one of claims 6 to 10 ,
In the plant, anthocyanidin pigments are accumulated in the cells of the petals of the plant in an amount of 30% by mass or more of the total amount of pigments present in the cells, and at least a portion of anthocyanidin modifying enzyme genes is deleted.
A part, tissue or cell of a plant.